This proposal constitutes a competitive renewal of a MERIT award research program to design and apply metallointercalators in targeting nucleic acids. Metal complexes have been designed that bind and react at DNA sites with high specificity either through shape-selection or through functionalization of ancillary ligands to obtain an ensemble of non-covalent contacts with DNA. We have also designed bulky rhodium intercalators that specifically target single base mismatches in the DNA duplex through photoactivated strand cleavage. We now propose the application of these metal complexes as new diagnostic agents and potentially as chemotherapeutic agents. Metal complexes with bulky intercalating ligands targeted to mismatches will be synthesized containing fluorophores, peptides, cleaving functionalities and metalation sites, notably Pt. Crystallographic and NMR studies of the complexes bound to oligonucleotides will be utilized to characterize metal-bound structures. Significantly, DNA mismatch-specific targeting agents will be applied to probe mismatch frequencies in mismatch repair (MMR) deficient versus proficient cell lines and to examine if mismatches are preferentially localized in different genes. Since MMR-deficiency is associated with cancerous transformation, these experiments form the basis for developing sensitive, direct diagnostic assays for cancerous transformations in tissues. Systematic studies with luminescent Rh and Ru complexes through both flow cytometry and confocal microscopy will be conducted to determine factors governing cellular uptake of the tris(metal) chelates, either functionalized or as more complex conjugates. With luminescent metal complexes targeted to mismatches, direct diagnostics of MMR-deficient cells are proposed. Given nuclear uptake results, metal complexes will also be examined in cytotoxicity and cell proliferation assays in MMR deficient versus proficient cell lines. By combining a DNA mismatch-targeting agent with a potent DNA damaging agent to preferentially damage MMR-deficient cells, those most susceptible to cancerous transformation, we plan to examine a completely new route to rationally designed anticancer agents.